A Validated Real-Time PCR Method for the Specific Identification of Probiotic Strain Lactobacillus rhamnosus GG (ATCC 53103)

Abstract Background Strain Lactobacillus rhamnosus GG is one of the best-studied and most widely used probiotic strains, with various health benefits. Because probiotic health benefits and safety are strain specific, the availability of a reliable assay for specific identification of Lactobacillus rhamnosus GG is vital to ensure probiotic efficacy. Objective To design and validate a probe-based real-time PCR assay for specific identification of strain Lactobacillus rhamnosus GG. Method Rapid Annotation using Subsystem Technology (RAST) was used to find a unique sequence region in the genome of Lactobacillus rhamnosus GG. A probe-based assay was designed and evaluated for specificity, sensitivity, efficiency, repeatability, and reproducibility. Results RAST identified a unique gene coding for a hypothetical protein in the genome of Lactobacillus rhamnosus GG. The assay successfully amplified all 22 target samples and did not amplify any of the 28 non-target strains, achieving 100% true positive and 0% false positive results. The Limit of Detection (LOD) was determined to be 0.001 ng. Reaction efficiency values, from three dilution series, were 96.4%, 93.3%, and 96.8% with R square values of 0.9974, 0.9981, and 0.9998, respectively. Relative standard deviation (RSD, %) of repeatability was below 1% and RSD of reproducibility was below 4%. Conclusions This Lactobacillus rhamnosus GG specific assay proved to be specific, sensitive, efficient, and reproducible. Since the assay was evaluated on two real-time PCR platforms, including a portable one, the assay can be used for onsite testing throughout the supply chain. Highlights The availability of validated and reliable assays for strain-specific identification plays a vital role in achieving compliance in probiotic products.

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Locked Nucleic Acid Hydrolysis Probes for the Specific Identification of Probiotic Strains Bifidobacterium animalis subsp. lactis DSM 15954 and Bi-07™

Frontiers in Microbiology ◽

10.3389/fmicb.2021.801795 ◽

2021 ◽

Vol 12 ◽

Author(s):

Hanan R. Shehata ◽

Anthony Kiefer ◽

Wesley Morovic ◽

Steven G. Newmaster

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Probiotic Strain ◽

Locked Nucleic Acid ◽

Identification Methods ◽

Bifidobacterium Animalis ◽

Pcr Assays ◽

Pcr Targeting

Probiotic health benefits are now well-recognized to be strain specific. Probiotic strain characterization and identification is thus important in clinical research and in the probiotic industry. This is becoming especially important with reports of probiotic products failing to meet the declared strain content, potentially compromising their efficacy. Availability of reliable identification methods is essential for strain authentication during discovery, evaluation and commercialization of a probiotic strain. This study aims to develop identification methods for strains Bifidobacterium animalis subsp. lactis DSM 15954 and Bi-07 (Bi-07™) based on real-time PCR, targeting single nucleotide polymorphisms (SNPs). The SNPs were targeted by PCR assays with locked nucleic acid (LNA) probes, which is a novel application in probiotic identification. The assays were then validated following the guidelines for validating qualitative real-time PCR assays. Each assay was evaluated for specificity against 22 non-target strains including closely related Bifidobacterium animalis subsp. lactis strains and were found to achieve 100% true positive and 0% false positive rates. To determine reaction sensitivity and efficiency, three standard curves were established for each strain. Reaction efficiency values were 86, 91, and 90% (R square values > 0.99), and 87, 84, and 86% (R square values > 0.98) for B. animalis subsp. lactis DSM 15954 and Bi-07 assays, respectively. The limit of detection (LOD) was 5.0 picograms and 0.5 picograms of DNA for DSM 15954 and Bi-07 assays, respectively. Each assay was evaluated for accuracy using five samples tested at three different DNA concentrations and both assays proved to be highly repeatable and reproducible. Standard deviation of Cq values between two replicates was always below 1.38 and below 1.68 for DSM 15954 and Bi-07 assays, respectively. The assays proved to be applicable to mono-strain and multi-strain samples as well as for samples in various matrices of foods or dietary supplement ingredients. Overall, the methods demonstrated high specificity, sensitivity, efficiency and precision and broad applicability to sample, matrix and machine types. These methods facilitate strain level identification of the highly monophyletic strains B. animalis subsp. lactis DSM 15954 and Bi-07 to ensure probiotic efficacy and provide a strategy to identify other closely related probiotics organisms.

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The Use of Real-Time Polymerase Chain Reaction Combined with Specific-Species Primer for Analysis of Dog Meat DNA in Meatball

Indonesian Journal of Chemistry ◽

10.22146/ijc.48930 ◽

2020 ◽

Vol 21 (1) ◽

pp. 225

Author(s):

Abdul Rohman ◽

Wiranti Sri Rahayu ◽

Sudjadi Sudjadi ◽

Sudibyo Martono

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Real Time Pcr ◽

Dna Analysis ◽

Limit Of Detection ◽

Specific Primer ◽

Chain Reaction ◽

Relative Standard ◽

Polymerase Chain ◽

Species Specific

The presence of dog meat is a crucial issue because dog meat is non-halal meat for Muslims. The objective of this study was to design and validate species-specific primer for the identification of dog meat DNA in meatball using real-time polymerase chain reaction (real-time PCR). The specific primer targeting mitochondrial cytochrome c oxidase subunit 1 (CO1) was validated. The specific primers used were designed using Integrated DNA Technologies (IDT) software and subjected to NCBI BLAST procedure. The candidate primers were tested for specificity study using several DNAs from fresh meat of pork, chicken, beef, lamb, and rat. The method was also validated by determining several parameters of linearity, sensitivity, precision, and efficiency. The results showed that primer could amplify specifically DNA target at an optimized annealing temperature of 56.6 °C. The limit of detection (LoD) obtained was 5 ng DNA, corresponding to 2.5% of dog meat in a meatball. The repeatability evaluation, expressed with relative standard deviation (RSD), and efficiency value was in the acceptable range (RSD < 25% and efficiency (90–105%). This method was successfully used for the analysis of marketed samples. Real-time PCR can be used as a standard method in halal authentication analysis through DNA analysis.

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Validation of a Novel Diagnostic Approach Combining the VersaTREK™ System for Recovery and Real-Time PCR for the Identification of Mycobacterium chimaera in Water Samples

Microorganisms ◽

10.3390/microorganisms9051031 ◽

2021 ◽

Vol 9 (5) ◽

pp. 1031

Author(s):

Roberto Zoccola ◽

Alessia Di Blasio ◽

Tiziana Bossotto ◽

Angela Pontei ◽

Maria Angelillo ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Detection System ◽

Bacterial Load ◽

Microbial Control ◽

Hospital Setting ◽

Sample Volume ◽

Analytical Sensitivity ◽

Initial Water

Mycobacterium chimaera is an emerging pathogen associated with endocarditis and vasculitis following cardiac surgery. Although it can take up to 6–8 weeks to culture on selective solid media, culture-based detection remains the gold standard for diagnosis, so more rapid methods are urgently needed. For the present study, we processed environmental M. chimaera infected simulates at volumes defined in international guidelines. Each preparation underwent real-time PCR; inoculates were placed in a VersaTREK™ automated microbial detection system and onto selective Middlebrook 7H11 agar plates. The validation tests showed that real-time PCR detected DNA up to a concentration of 10 ng/µL. A comparison of the isolation tests showed that the PCR method detected DNA in a dilution of ×102 CFU/mL in the bacterial suspensions, whereas the limit of detection in the VersaTREK™ was <10 CFU/mL. Within less than 3 days, the VersaTREK™ detected an initial bacterial load of 100 CFU. The detection limit did not seem to be influenced by NaOH decontamination or the initial water sample volume; analytical sensitivity was 1.5 × 102 CFU/mL; positivity was determined in under 15 days. VersaTREK™ can expedite mycobacterial growth in a culture. When combined with PCR, it can increase the overall recovery of mycobacteria in environmental samples, making it potentially applicable for microbial control in the hospital setting and also in environments with low levels of contamination by viable mycobacteria.

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Quantitative Detection of Clostridium perfringens in the Broiler Fowl Gastrointestinal Tract by Real-Time PCR

Applied and Environmental Microbiology ◽

10.1128/aem.71.7.3911-3916.2005 ◽

2005 ◽

Vol 71 (7) ◽

pp. 3911-3916 ◽

Cited By ~ 71

Author(s):

Mark G. Wise ◽

Gregory R. Siragusa

Keyword(s):

Gastrointestinal Tract ◽

Real Time ◽

Clostridium Perfringens ◽

Real Time Pcr ◽

Limit Of Detection ◽

Quantitative Detection ◽

Necrotic Enteritis ◽

Analytical Sensitivity ◽

Quantitative Real Time Pcr ◽

Pcr Inhibitor

ABSTRACT Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 102 CFU/g of ileal material, but only about 104 CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.

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Rapid Species Identification of Cooked Poisonous Mushrooms by Using Real-Time PCR

Applied and Environmental Microbiology ◽

10.1128/aem.02082-07 ◽

2008 ◽

Vol 74 (10) ◽

pp. 3306-3309 ◽

Cited By ~ 24

Author(s):

Kazuhiko Maeta ◽

Tomoya Ochi ◽

Keisuke Tokimoto ◽

Norihiro Shimomura ◽

Nitaro Maekawa ◽

...

Keyword(s):

Real Time ◽

Species Identification ◽

Real Time Pcr ◽

Specific Test ◽

Specific Identification ◽

Specific Fluorescence ◽

Species Specific ◽

Poisonous Mushrooms

ABSTRACT Species-specific identification of the major cooked and fresh poisonous mushrooms in Japan was performed using a real-time PCR system. Specific fluorescence signals were detected, and no nonspecific signals were detected. Therefore, we succeeded in developing a species-specific test for the identification of poisonous mushrooms within 1.5 h.

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Development and validation of a triplex real-time PCR for rapid detection and specific identification of M. avium sub sp. paratuberculosis in faecal samples

Veterinary Microbiology ◽

10.1016/j.vetmic.2008.09.087 ◽

2009 ◽

Vol 136 (1-2) ◽

pp. 166-172 ◽

Cited By ~ 30

Author(s):

Léonid M. Irenge ◽

Karl Walravens ◽

Marc Govaerts ◽

Jacques Godfroid ◽

Valérie Rosseels ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Rapid Detection ◽

Specific Identification ◽

Faecal Samples ◽

Development And Validation

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Quantitative Real-Time PCR Assay Panel for Detection and Type-Specific Identification of Epidemic Respiratory Human Adenoviruses

Journal of Clinical Microbiology ◽

10.1128/jcm.03297-12 ◽

2013 ◽

Vol 51 (4) ◽

pp. 1089-1093 ◽

Cited By ~ 26

Author(s):

X. Lu ◽

E. Trujillo-Lopez ◽

L. Lott ◽

D. D. Erdman

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Quantitative Real Time Pcr ◽

Pcr Assay ◽

Specific Identification ◽

Human Adenoviruses

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Development and evaluation of duplex TaqMan real-time PCR assay for detection and differentiation of wide-type and MGF505-2R gene-deleted African swine fever viruses

10.21203/rs.3.rs-59710/v2 ◽

2020 ◽

Author(s):

zhenhua Guo ◽

Kunpeng Li ◽

Songlin Qiao ◽

Xinxin Chen ◽

Ruiguang Deng ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Large Scale ◽

Limit Of Detection ◽

African Swine Fever ◽

Economic Losses ◽

Clinical Samples ◽

Diagnosis Method ◽

Pcr Method ◽

And Control

Abstract Background: African swine fever (ASF) is the most important disease to the pigs and cause serious economic losses to the countries with large-scale swine production. Vaccines are recognized as the most useful tool to prevent and control ASF virus (ASFV) infection. Currently, the MGF505 and MGF360 gene-deleted ASFVs or combined with CD2v deletion were confirmed to be the most promising vaccine candidates. Thus, it is essential to develop a diagnosis method to discriminate wide-type strain from the vaccines used.Results: In this study, we established a duplex TaqMan real-time PCR based on the B646L gene and MGF505-2R gene. The sequence alignment showed that the targeted regions of primers and probes are highly conserved in the genotype II ASFVs. The duplex real-time assay can specifically detect B646L and MGF505-2R gene single or simultaneously without cross-reaction with other porcine viruses tested. The limit of detection was 5.8 copies and 3.0 copies for the standard plasmids containing B646L and MGF505-2R genes, respectively. Clinical samples were tested in parallel by duplex real-time PCR and a commercial ASFV detection kit. The detection results of these two assays against B646L gene were well consistent.Conclusion: We successfully developed and evaluated a duplex TaqMan real-time PCR method which can effectively distinguish the wide type and MGF505 gene-deleted ASFVs. It would be a useful tool for the clinical diagnosis and control of ASF.

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Direct detection of microorganisms responsible for the sexually transmitted infection on the mobile real-time PCR device without treating in advance

10.1101/2021.03.02.21252726 ◽

2021 ◽

Author(s):

Masaaki Muraoka ◽

Kazunori Sohma ◽

Osamu Kawaguchi ◽

Mikio Mizukoshi

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Direct Detection ◽

Treponema Pallidum ◽

Nucleic Acid Amplification ◽

Direct Pcr ◽

Transmitted Infection ◽

Sexually Transmitted ◽

Ct Value

ABSTRACTAs WHO reported, four curable STIs-chlamydia, gonorrhoea, syphilis and trichomoniasis occur more than 1 million per each day globally almond 2016. For this reason, it is important to control these STIs, one of which is “to detect”. The general methods in order to detect STIs are nucleic acid amplification tests (NAATs). One of the reasons why NAATs are utilized in many tests is that it is possibly to be more sensitive than other test. However, there needs to treat extraction of nucleic acids in advance and amplify specific regions by NAATs, and hence it must take much labour and much time. In this work, for Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Treponema pallidum (TP) which is each etiological agent of chlamydia, gonorrhoea and syphilis, we evaluate and propose “quicker and simpler” NAATs. Specifically, utilizing mobile real-time PCR device “PCR1100” and PCR reagent kit “KAPA3G Plant PCR Kit”, it was considered whether real-time direct PCR could be performed or not without treating DNA extraction in advance so-called “direct”.As a result, firstly, we established that real-time direct PCR could be performed in all of CT, NG, and TP, and moreover, each Ct value correlated with the concentration of each organism similarly to detection of genome DNA (each correlation coefficient R2 > 0.95). Moreover, each assay demonstrated a limit of detection (LOD) of the follows; CT was 10^0.86 = 7.24 IFU/reaction, NG was 10^-0.19 = 0.65 CFU/reaction, and TP was 10^1.4 = 25.1 organisms/reaction. However, it appeared the sensitivity was a little low, especially for CT and TP.Secondly, we found that even as without treating sample in advance, the time of detection was required more less 15 minutes at any of case, which was very quick compared with other current methods for real-time PCR. Additionally, compared with other commercial devices, it was easier to operate the PCR1100 device, for example, start, analysis of Ct value.In conclusion, the present study has demonstrated that it is possible for real-time direct PCR to perform with combination of the PCR1100 device and the PCR reagent kit in 3 kinds of microorganisms-CT, NG and TP. Furthermore, we propose “quicker and simpler” methods for NAATs, which it would not take labour and time. Further studies are needed in order to contribute to control STIs.

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